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Pergamon PII] S99208311"86#909113

Phytochemistry\ Vol[ 38\ No[ 4\ pp[ 02660279\ 0887 0887 Elsevier Science Ltd[ All rights reserved Printed in Great Britain 99208311:87:,*see front matter

TOXIC AMINES AND ALKALOIDS FROM ACACIA RIGIDULA

BEVERLY A[ CLEMENT\ CHRISTINA M[ GOFF and T[ DAVID A[ FORBES$
Department of Veterinary Anatomy and Public Health\ Texas A + M University\ College Station\ TX 66732!3347^ $ Texas A + M University Agricultural Research and Extension Center\ Uvalde\ TX 67790\ U[S[A[ "Received 09 June 0886#

Key Word Index*Acacia ri`udula^ blackbrush^ Leguminosae^ alkaloids^ GC:MS[

Abstract*Acacia ri`idula Benth[\ blackbrush\ is a shrub found growing on rocky ridges in west and southwest Texas and northern Mexico[ Consumption of blackbrush and a related species guajillo\ Acacia berlandieri Benth[\ has been associated with a locomotor ataxia known as {{limber leg||[ In an e}ort to identify the mechanism of this toxicity\ blackbrush was subjected to rigorous chemical analysis[ In addition to the four previously detected amines\ N!methyl!b!phenethylamine\ tyramine\ N!methyltyramine\ and hordenine\ 39 other alkaloids and amines were isolated and identi_ed by GC!MS[ These alkaloids and amines included nicotine\ N\N!dimethyltryptamine\ mescaline\ several tetrahydroisoquinoline alkaloids\ and four amphetamines[ A sig! ni_cant increase in the number and relative quantities of these compounds was observed in late season foliage[ 0887 Elsevier Science Ltd[ All rights reserved

INTRODUCTION

Acacia ri`udula Benth[\ blackbrush\ is a shrub found growing on rocky ridges in southwest and west Texas\ and in the northern states of Mexico\ including Tamaulipas\ Nuevo Leon\ Chihuahua\ San Luis Potosi\ and Jalisco 0[ The leaves are bipinnate and predominately short 0414 mm long\ pinnae 01 pairs that are 504 mm long\ with 13 pairs "rarely 4 pairs# of elliptic to oblong lea~ets 301 mm long and 57 mm wide[ Blackbrush is heavily defended with long sharp spines and\ if left unchecked\ will form virtually impenetrable thickets[ In addition to its physical defences\ A[ ri`idula has been shown to contain appreciable levels of toxic alkaloids[ Sheep and goats grazing on a related species\ Acacia berlandieri Benth[\ guajillo\ during periods of drought in the Rio Grande Plains of Texas have developed a locomotor ataxia referred to as {{guajillo wobbles|| or {{limber leg|| 1[ Previous analysis of blackbrush had detected and identi_ed four amines\ Nmethyl!b!phenethylamine "NMPEA#\ tyramine\ N!methyltyramine\ and hord! enine\ which were also found in guajillo 2[ The locomotor ataxia developed in the early A[ berlandieri feeding trials 3 was not observed in adequately fed animals injected with NMPEA or tyr! amine 4[ A review 5 of previous work has shown that many plant species endemic to southwest Texas and northern Mexico contain a wide variety of aro!

matic monoamines[ A need to more fully explain the results of earlier studies\ together with advances in GC!MS technology\ led to an intensive chemical analysis of A[ berlandieri to identify other amines and alkaloids present in the leaves 6[ As a result of this study\ an intensive chemical analysis of A[ ri`idula Benth[ was undertaken to identify the amines and alkaloids present in the leaves and stems that would comprise browse material[

RESULTS AND DISCUSSION

Author to whom correspondence should be addressed[

Leaves and stems that would comprise browse material were packed into an extraction thimble\ placed in a Soxhlet apparatus and extracted with methanol followed by extraction with chloroform[ This extraction procedure was compared with the more traditional methods of extraction by soaking the plant material in acid solution 1\ 7 and found to produce a cleaner\ more complete isolation of the amines and alkaloids present in the sample[ The initial extract was fractionated by extraction with aq[ acid\ the pH of the acid extract was adjusted to ca pH 09 prior to re!isolation of the alkaloids by extraction with organic solvents[ The pH of the aq[ fraction was routinely checked between extractions and adjusted "dil[ NaOH added# as necessary[ It was found that when the pH exceeded 00\ a large portion of the phenol containing alkaloids remained in the aqueous solution as their corresponding phenolate salts[ Particular care was taken to maintain the extracts and isolates under

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B[ A[ CLEMENT et al[

an inert atmosphere[ Left unprotected\ the isolated amines and alkaloids readily decomposed[ Forty!four amines and alkaloids\ including the four previously encountered amines\ N!methyl!b!phene! thylamine\ tyramine\ N!methyltyramine\ and hord! enine\ were identi_ed by GC!MS[ Both splitless injec! tion and dedicated on!column injection systems were employed for GC!MS analysis[ The on!column injec! tion is a much milder method of sample introduction[ This technique\ although it produced the most com! plex chromatograms\ was best for the detection of the phenol containing components of the mixture which tended to be more thermally labile[ No derivatization was performed on the analytes\ therefore GC!MS analysis would only be expected to detect the volatile amines and alkaloids present in the sample[ The majority of the isolated alkaloids\ 18 of the 33 identi_ed\ were related to the parent compound b! phenethylamine[ These compounds generally varied in the degree of N!methylation\ a!methylation "amphetamine family#\ and in oxygenation of the aro! matic ring "tyramine\ dopamine\ and mescaline famil! ies#[ The 1!cyclohexylethylamine and the N!cyclohex! ylethyl!N!methylamine are the saturated analogs of the phenethylamine and NMPEA respectively[ Tryp! tamine\ N!methyltryptamine\ and N\N!dime! thyltryptamine were also isolated from blackbrush[ Tryptamine and N\N!dimethyltryptamine were also detected in the related species guajillo 6\ albeit at relatively low levels[ Early season levels of tryptamine in A[ berlandieri range from 89 to 013 ppb while late season levels range between 176 and 223 ppb[ N\N! Dimethyltryptamine was only detected in late season guajillo at levels ranging between 64 and 004 ppb[ It is interesting to note that a similar animal ataxia called {{Phalaris staggers|| occurs in Australia and is associ! ated with consumption of various Phalaris species which are known to contain N\N!dimethyltryptamine 8[ Other noteworthy alkaloids found in blackbrush include nicotine\ nornicotine\ and four tetrah! ydroisoquinoline alkaloids\ anhalamine\ anhalidine\ anhalonidine\ and peyophorine[ The amides of the amino acids pipecolic acid and p!hydroxypipecolic acid were also detected[ It should be noted that no other amino acid esters or amides were detected in the extract[ Initial identi_cation of the alkaloids present within the plant extracts was based upon library comparison of their MS fragmentation patterns with the _nal con! _rmation of identi_cation made by direct comparison with spectra of authentic samples[ Quanti_cation of the levels of each compound was based upon standard curves generated with authentic samples[ Leaves\ peti! oles and unligni_ed stems were collected twice\ a _rst growth sample collected early in the spring and a late season sample collected before frost in the autumn[ As was previously found with NMPEA the foliage collected in the autumn contained higher quantities of amines and alkaloids 09[ There was also a distinct

increase in the number and quantity of methylated analogs present "Table 0#[ Several as yet unidenti_ed amine!containing com! pounds have been detected but have not yet been identi_ed[ It is also probable that several biosynthetic precursors are present in the mixture but as yet are unresolved and are below the threshold of GC!MS detection[ Phenolic amines\ as a group\ impact the hypo! thalamicpituitaryadrenal axis 00[ The consequent release of ACTH and cortisol results in sym! phathomimetic action[ The number of phenolic amines reported in Table 0 and their concentrations in the plant indicate a substantial toxic load to animals consuming blackbrush[ The toxicity of nicotine and nornicotine has been well established 01\ as has the psychoactivity of mescaline and its derivatives[ None of the compounds identi_ed appear to have been implicated in locomotor ataxia[ However the presence of the amphetamines suggests the possibility for a reduction of monoamine oxidase activity 02[
EXPERIMENTAL

GC!quadrupole MS "EIMS at 69 eV# were obtained on Hewlett Packard Model 4877A and Model 4869C systems each having both splitless heated injection port and dedicated on!column injection capabilities[ Grade 9 He was used as the carrier gas and the transfer line was maintained at 179>[ Heated injection con! ditions include] head pressure 4 psi^ injection volume 0 ml^ injection port 199>^ splitless time 0 min^ purge ~ow 59 ml:min^ initial time 0 min^ ramp 2>:min^ _nal temp[ 169>^ _nal time 19 min[ On!column injection conditions include] head pressure 4 psi^ injection vol! ume 0 ml^ initial temp[ 59>^ initial time 0 min^ ramp 0[4>:min^ _nal temp[ 169>^ _nal time 24 min[ WCOT cross!linked methyl silicone 01 "only used on HP 4869 system# and 25 m\ 9[1 mm i[d[\ 22 m coating in thick! ness capillary columns were used[ Mass spectral data was collected as the total ion chromatograms in the operating range of 24799 amu[ The GC!MS opera! ting system including the Wiley mass spectral library and the NBS mass spectral library of standards[ Pre! liminary identi_cation was made by library compari! son\ _nal identi_cation was made by direct spectral comparison with the spectra of an authentic sample obtained from the GC!MS[ Authentic samples were either purchased or prepared by known chemical pro! cedures[ GC!MS quanti_cation was made by using the area under a selected m:z peak for each compound and comparing this to a standard curve generated by injec! tion of a series of known standards[ Care was taken to make sure that the detector response was linear in the concentration range being run[ Collection of leaves and stems Samples of A[ ri`idula Benth[ were collected from plants growing on a southwest!facing slope in Zavala

Toxic amines and alkaloids from Acacia ri`idula Table 0[ Amines and alkaloids from A[ ridi`ula Benth[ Early season "ppm# 1!Cyclohexylethylamine N!1!Cyclohexylethyl!N!methylamine Phenethylamine N!Methylphenethylamine N\N!Dimethylphenethylamine Amphetamine Methamphetamine N\N!Dimethyl!a!methylphenethylamine p!Hydroxyamphetamine p!methoxyamphetamine Tyramine N!Methyltyramine Hordenine "anhaline# Dopamine N!Methyldopamine N\N!Dimethyldopamine 2!Methoxytyramine N!Methyl!2!methoxytyramine 2!Hydroxy!3!methoxyphenethylamine N!Methyl!2!hydroxy!3!methyoxyphenethylamine 2\3!Dimethoxyphenethylamine N!Methyl!2\3!dimethoxyphenethylamine 2\3\4!Trihydroxyphenethylamine N!Methyl!2\3\4!trihydroxyphenethylamine Mescaline N!Methylmescaline Trichocereine 2\4!Dimethoxytyramine 2\3!Dimethoxy!4!hydroxy!b!phenethylamine b!Methoxy!2\3!dihydroxy!4!methoxy!b!phenethylamine 2\3!Dimethoxy!a!methyl!4!hydroxy!b!phenethylamine Tryptamine N!Methyltryptamine N\N!Dimethyltryptamine Nicotine Nornicotine Anhalamine Anhalidine "N!methylanhalamine# Anhalonidine Peyophorine Pipecolamide p!Hydroxypipecolamide 0\3!Benzezediamine 3!Methyl!1!pyridinamine 9[7 0[1 761[2 1203[5 012[5 5[6 nd 46[5 1[0 nd 348[0 126[3 5[3 7[8 9[4 00[1 0[7 2[3 04[7 08[1 0[2 6[5 0[5 9[2 2[3 0[7 9[1 0[5 04[5 3[5 4[2 9[7 3[5 212[7 34[7 12[3 8[5 4[5 1[2 2[7 761[7 130[5 093[7 230[4 Late season "ppm# 24[1 36[0 0024[6 4153[7 613[4 00[7 01[3 283[1 5[8 04[6 0588[1 0126[5 422[7 25[0 7[1 33[5 01[8 17[3 052[1 073[6 5[4 17[2 01[3 0[8 16[4 24[2 02[7 10[5 46[0 11[0 50[3 10[1 43[8 457[3 041[3 73[2 37[6 40[1 04[6 32[3 867[1 242[0 018[5 456[2

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County\ Texas[ Early season collection was performed in the spring after vigorous new growth appeared[ Late season collection was performed in the late fall prior to the _rst front and before color change was detected[ Leaves\ petioles\ and attached tender stems were collected until ca 499 g of fr[ wt had been gath! ered[ The material was sealed in waterproof bags and placed on ice immediately[ Samples were then frozen at 19> and stored until extraction[ Voucher speci! mens were collected and stored at the Texas A + M University Agricultural Research and Extension Center\ Uvalde\ Texas[

Soxhlet extraction procedure Still frozen A[ ri`idula was packed into Whatman single thickness cellulose extraction thimbles "ca 49 g:thimble\ 199 g total# and extracted continuously for 13 h with MeOH[ The MeOH was removed and replaced with CHCl2 and the extraction continued for an additional 13 h[ A pilot study established that the extracts could be safely concentrated by rotary evaporation "water aspirator#[ The MeOH extract was concentrated and the residue dissolved in 099 ml of CHCl2[ This solution was extracted 2 with 49 ml

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portions of 09) aq[ HCl[ The acid fractions were combined\ the pH adjusted to ca 09[2 by addition of aq[ NaOH\ and the resulting solution was _rst extracted 2 with 49 ml portions of CHCl2 followed by extraction 2 with 49 ml portions of EtOAc[ These organic extracts were combined\ dried with MgSO3\ _ltered\ concentrated under vacuum\ and stored under argon prior to analysis by GC!MS[ The CHCl2 frac! tion from the Soxhlet extraction was handled in an identical fashion to the MeOH extract with the excep! tion that it was not _rst concentrated prior to aq[ acid extraction[ The MeOH and CHCl2 extracts from the Soxhlet extraction were not combined[ Acid extraction of plant material Acacia ri`idula\ 099 g\ was placed into a 0 l Erlen! meyer ~ask and mixed with 499 ml of 09) aq[ acid "HCl and HOAc were both used#[ The suspension was stirred under argon at 59> overnight[ The darkened suspension was _ltered through glass wool covered with a 1 cm bed of sand[ The _ltrate was extracted 1 with 099 ml portions of EtOAc followed by 2 extraction with 099 ml portions of CHCl2[ The pH of the _ltrate was adjusted to ca 09 by addition of NaOH pellets[ This solution was extracted 2 with 049 ml portions of CHCl2 followed by extraction 2 with 049 ml portions of EtOAc[ The organic extracts were combined\ dried over MgSO3\ _ltered\ concentrated under vacuum and stored under argon prior to analy! sis by GC!MS[ Acknowled`ments*We thank the Texas Advanced

Technology Program Grant 909255!042 for partial support of this project[

REFERENCES

0[ Vines\ R[ A[\ Trees\ Shrubs\ and Woody Vines of the Southwest[ University of Texas Press\ Austin\ TX\ 0873[ 1[ Price\ D[ A[ and Hardy\ W[ T[\ J[ Am[ Vet[ Med[ Assoc[\ 0842\ 15\ 111[ 2[ Adams\ H[ R[ and Camp\ B[ J[\ Toxicon\ 0855\ 3\ 74[ 3[ Broughton\ I[ B[ and Hardy\ W[ T[\ 43th Ann[ Rep[\ Texas Agric[ Exper[ Stat[\ 0830\ p[ 048[ 4[ Forbes\ T[ D[ A[\ Randel\ R[ D[\ Tolleson\ D[ R[ and Hensarling\ C[ M[\ S[ Afric[ J[ Anim[ Sci[\ 0882\ 12\ 085[ 5[ Smith\ T[ A[\ Phytochemistry\ 0866\ 05\ 8[ 6[ Clement\ B[ A[\ Go}\ C[ M[ and Forbes\ T[ D[ A[\ Phytochemistry\ 0886\ "in press#[ 7[ Camp\ B[ J[ and Moore\ J[ A[\ J[ Am[ Pharm[ Assoc[\ 0885\ 38\ 047[ 8[ Lee\ H[ J[ and Kuchel\ R[ E[\ Austral[ J[ A`ric[ Res[\ 0842\ 3\ 77[ 09[ Forbes\ T[ D[ A[\ Pemberton\ I[ J[\ Smith\ G[ R[\ Hensarling\ C[ M[ and Tolleson\ D[ R[\ Texas A`ricultural Experiment Station Consolidated Pro`ress Report No[ 3764\ 0880[ 00[ Vera!Avila\ H[ R[\ Randel\ R[ D[ and Forbes\ T[ D[ A[\ Dom[ Anim[ Endocrinol[\ 0885\ 02\ 174[ 01[ Duke\ J[ A[\ Handbook of Medicinal Herbs[ CRC Press\ Boca Raton\ FL[ 02[ Mantle\ T[ J[\ Tiptons\ K[ F[ and Garret\ N[ J[\ Biochem[ Pharm[\ 0865\ 14\ 1962[